Native collagen has a primary structure of repeating trimeric amino acid sequences. Within a helical region, which constitutes about 95% of the molecule, the amino acid glycine (Gly) occurs at every third position of a peptide trimer. Imino residues (I), either proline (Pro) and hydroxyproline (Hyp), occur in 56% of the trimers, 20% as Gly-X-I; 27% as Gly-I-Y; and 9% as Gly-I-I. Pro usually occurs in the second position in the repeating trimer; while Hyp usually occurs in the last position. (Bhatnagar, R. and R. Rapaka (1976) Chap. 10 in Biochemistry of Collagen R. Ramachandren, ed. Plenum Press, New York. pp.481-482).
Tripeptide sequences (Gly-X-Y) wherein X and Y are amino acid residues other than proline (Pro) or hydroxyproline (Hyp) make up 44% of the collagen amino acid trimers. Glutamic acid, leucine, and phenylalanine occur mostly in the X position and threonine, glutamine, methionine, arginine and lysine occur mostly at the Y position. With the exception of alanine and serine, the X position amino acids have bulky side chains.
Synthetic collagens are of interest because they provide materials for collagen-like biomaterials having diverse clinical applications, including use in drug delivery devices, ocular devices, and wound healing materials. Because the proline and hydroxyproline (a post-translationally modified proline residue) residues are abundant in natural collagen sequences, many sequential polymers composed of the trimeric amino acid sequences Gly-Pro-Xaa and Gly-Xaa-Pro (where Xaa is any natural amino acid residue) have been prepared to mimic the collagen structures. (Segal, D. M., and Traub, W.(1969) J. Mol. Biol. 43:487-496 disclose poly(L-alanyl-L-prolyl-glycine); Segal, D. (1969) J. Mol. Biol. 43:497-517) discloses collagen-like polyhexapeptides (Gly-Ala-Pro-Gly-Pro-Pro).sub.n, (Gly-Pro-Ala-Gly-Pro-Pro).sub.n, Gly-Ala-Pro-Gly-Pro-Ala).sub.n, and (Gly-Ala-Ala-Gly-Pro-Pro).sub.n ; Sakakibara, S. (1973) Biochim. Biophys. Acta 303:198-202 discloses (Pro-Hyp-Gly).sub.n ; Scatturin, A. (1975) Intl. J. Peptide Protein Res. 7:425-435 discloses (Pro-Leu-Gly).sub.n, and (Leu-Pro-Gly).sub.n ; Bansal, M. (1978) Peptide Protein Res. 11:73-81 discloses (Gly-Pro-Leu).sub.n and (Gly-Leu-Pro).sub.n ; and Miller, M. (1980) Macromolecules 13:910-913) discloses poly(glycylprolylalanyl). Ananthanarayanan, V. et al. (1976) in Chap. 15, Biopolymers, pp. 707-716 (J. Wiley & Sons) disclose polymers wherein the triplet contains the isomeric N-methyl glycine sarcosine as (Gly-Pro-Sar).sub.n and (Gly-Sar-Pro).sub.n. The publications cited above are incorporated by reference.
Collagen has a characteristic tertiary, secondary and primary structure. Most polypeptides comprise sequences of amino acids in peptide linkage which are arrayed either in an .alpha.-helix, a right-handed spiral, or alternatively, in a pleated sheet .beta.-conformation. In each of these arrays the amino acids of neighboring polypeptide strands are held in place by intramolecular hydrogen bonds. Collagen, by comparison, is made up of three polypeptide chains comprising repeating amino acid trimers. These chains are arrayed in three extended left handed spirals of about three residues per turn, the polyproline II-like chains (Rich, A. et al. (1955) Nature 176:915). The polyproline II-like chains of collagen are arranged in a parallel direction and intertwined to adopt a supercoiled, or coiled coil, right-handed triple helix conformation (Bella, J. et al. (1994) Science 266:75-81) that is also characteristic of collagen. The chains that make up the collagen triple helix can be homotrimeric, that is, made up of identical repeating amino acid trimers, or they can be heterotrimeric, made up of chains of different amino acid trimers.
The association of polyproline II-like chains into a triple helix occurs spontaneously; however, the rate of helix formation may be slow because of the repulsive like charges at the amino and carboxyl ends of the polypeptide sequences that oppose an association of the chains. This "end effect" becomes less important as chain length increases. The rate of helix formation can also be slow because the amino acids in each chain must first line up, or be in "register" properly with each other, and to do so they must adjust position appropriately, one along the length of the other. Collagen chains have been found to require a one residue shift between corresponding amino acids in each chain in order to register properly and form trans amides for all peptide bonds.
Mimicry of natural collagen structures has been directed to enhancing their biostability by inserting unnatural residues into the peptide sequences. To enhance the biostability of collagen-like structures therefore, many unnatural proline analogs and other unnatural imino acid residues have been used to replace the frequently occurring proline residue in the peptide sequences. However, incorporation of such residues, such as the lower homologue of proline, azetidine-2-carboxylic acid (Aze), has been found to destabilize the triple helical structure of collagen or to prevent its formation (Zagari, A. et al. (1994) Biopolymers, 34:51-60).
Peptoid residues are a new class of unnatural imino acids (Simon, R. J. et al. (1992) Proc. Natl. Acad. Sci. USA 89:9367-9371) containing N-substituted glycine residues wherein the substituents on the nitrogen atom are the .alpha.-position side chains of amino acids. Because they are amino acids that do not occur in nature, peptoid residues or peptides containing peptoid residues have higher resistance to enzymatic attacks. In recent years, peptoid residues have been widely used in the design and synthesis of drugs and other peptide related biomaterials.
Template directed synthesis, the interaction of one molecule with an assembly of atoms to induce a preferred molecular architecture, is known in nature. The replication of DNA, for example, involves a templated synthesis of daughter polynucleotides from progenitor molecules having the same tertiary structure. A template-assembling approach has been widely used for the design and synthesis of protein analogs with high molecular weights. Molecular templates have been used in chemical synthesis to fix peptide loops and to induce the .alpha.-helix and .beta.-turn structures of polypeptides. Kelly, T. R. et al. (1990) J. Amer. Chem. Soc. 112:8024-8034, have reported use of a linear template to form .beta.-structures. Muller discloses anthracene-type tricyclic structures that can bridge two antiparallel peptide .beta.-strands or induce .beta.-turns. Muller also discloses that Kemp triacid condensed with glycine or alanine can act as a templates for inducing .alpha.-helicity of an attached polypeptide (Muller, K. et al. (1993) Chap. 33 in Perspectives in Medicinal Chemistry, B. Testa et al., eds, Verlag, Basel). Ghadiri, M. et al. (1993) Angew. Chem. Int. Ed. Engl. 32:1594-1597 have prepared a polypeptide 3-.alpha.-helix bundle containing a ruthenium metal bipyridyl complex.
Roth, W. et al. (1980) Biopolymers 19:1909-1917 has used lysine dimers and 1,2,3-propane carboxylic acid to prepare covalently bridged synthetic collagen model peptides which were found to assemble into a triple helix. Fields, C. G. et al. (1993) Biopolymers 33:1695-1707, and Tanaka, T. et al. (1993) FEBS 13257 334 (3) :272-276, have used two consecutively connected lysine residues with three functional amino groups to link three peptide chains at the C-termini or the N-termini. Fields et al. report the formation of thermally stable collagen-like polypeptide sequences in triple helical conformation by a solid phase procedure wherein three collagen-like peptide strands were synthesized in parallel from an origin at adjacent amine groups at the C-terminal. The branching of the peptide chains is reported to ensure the proper alignment or register of the chains in the triple helical polypeptide as they would be in native collagen.
In order to prepare synthetic collagen that has the properties of native collagen, the synthetic material must mimic collagen in tertiary as well as primary and secondary structure.